Metagenomic Analysis of Human Breast Milk
A study by Ward T.L. et al. assessed the diverse microbial community in human breast milk by a metagenomic analysis called Illumina sequencing. They found that the human milk contained over 320 prokaryotic genera. The most prominent phyla in the breast milk was the Proteobacteia, which totaled 65% of the bacterial community 1. Human Breast Milk Human breast milk has many nutrients for an infant child. It includes things like pre-biotics and immune proteins. Bacteria in the breast milk has a symbiotic and mutualistic relationship with the child's gastrointestinal tract. The bacteria that colonizes in the gut prevents the colonization of pathogenic bacteria, helps the development of mucosal cells in the gut, as well as enhances the development of the child's immune system 1. Recent methods to determine the microbiome of breast milk includes the amplification of 16S ribosomal RNA genes and then pyrosequencing of the rRNA. However, it does not allow one to determine how the microbes in the milk function and doesn't allow us to know which DNA motifs the infant is exposed to 1. In this study uses Illumina sequencing and the MG-RAST pipeline to determine the metagenome of the human breast milk. By using these methods the scientists could not only determine the type of bacteria in the beast milk but they could search for DNA motifs that modulate the immune system of the infant as well as determine which open reading frames influence the bacteria in the GI tract and maintain the stability of the GI tract 1. Results and Discussion Using the Ilumina metagenome sequencing method 261,532,204 sequences wer e read from the human breast milk. Of that 1,331,996 51bp sequences were of known prokaryotic genomes. 75% matched up to Staphylococcus sequences, 15% matched up to Pseudomonas, 2% matched Edmardsiella, and finally 1% matched to Pantoea, Treponema, Streptococcus, and Campylobacter. The final 3 % of the prokaryotic sequences match 361 bacterial genera 1. There were 74,189,220 sequences (Figure 1) that did not match the human genome or the prokaryotic genome. Some of these were re-aligned to the human genome with a <2 bp mismatch leaving 32,991,450 sequences to be aligned into contigs. This contigs were then submitted to the MG-RAST pipeline program. Of those sequences 53,785 made it through quality control and had a length of about 160 bp. The MG-RAST found that 65.1% of the contains aligned with the Proteobacteria phyla, and 34.6% aligned with the Firmicutes phyla. The contains also aligned to 194 known genomes. The highest being the Pseudomonas at 61.1% and the second highest being the Staphylococcus at 33.4% 1. This study also compared the contains from the breast milk to the feces of infants beast-fed, infants formula-fed and mothers. This found that the amount of Proteobacteria in the human milks metagenome was most similar to the feces of the breast-fed infant. Figure 2 shows how the contents of the breast milk compares to the breast-fed infant feces, the formula-fed infant feces and the mothers feces 1. The functional open reading frames of the breast milk were also compared to the fecal samples. They found that the fecal sample from the formula-fed infants was the least similar to the berate milk because 17 of the the 26 functional open reading frames were different between the two samples. Figure 3 shows the open reading frames present in each of the samples and they were each assigned to a functional categories 1. The contigs in the human breast milk and the fecal samples were searched for the presents of immunosuppressive motifs. The occurrence of the motifs ( TCAAGCTTGA and TTAGGG) were higher in the breast milk then in the fecal samples (Figure 4). The fecal sample of the breast-fed infants, formulated infants, and mothers were all very similar to the breast milk which suggests that the diversity of the microbes may lead to a diverse amount of immunosuppressive motifs 1. The motif TTAGGG was higher in the human genome then in the bacterial genome ad are mostly made of macrophages, neutrophils, and natural kill cells. This could mean that ingesting the mothers' DNA can lead to immune protection for the infant during development because of the balance of immune stimulation by bacterial CpGs and immune suppression via the mothers genome and well and the bacterial genome 1. Conclusion In this study 10 human milk samples were pooled together and sequence using the Illumia sequencing methods. The metagenomic sequencing of the breast milk found that there were over 56,000 contigs, which aligned to 177 bacterial genera. The reason the study compared the metagenome of breast milk to the feces of infants and mothers is because it give a good idea of how breast milk leads to the proper colonization of the infant gut. the breast milk microbiota was more similar to the breast-fed infants when it came to the amount of Proteobacteria colonies. However, the feces of the mothers and the breast milk were similar in their amounts of Firmicutes (59.5% and 34.6% respectively), which correlated with the hypothesis that immune cells with bacteria from the mothers gut are being carried to the breast milk for the infant child 1. This study also found that within the breast milk there is an abundance of DNA motifs within the bacteria and human DNA collected that are immunosuppressive. They also found that there are open reading frames within the milk that allow that bacteria in the breast milk metagenome to survive. This two finding demonstrate how we can begin to understand human breast milk at a functional level not only a phylogenetic level. Further study should be done to analysis how the function of the breast milk can relate to a infants health outcomes 1. References 1. Ward T.L., Hosid S., Ioshikhes I., Altosaar I. (2013) Human milk metagenome: a functional capacity analysis. BMC Microbiology 13:116. Link: http://www.ncbi.nlm.nih.gov.ezproxy.uvm.edu/pmc/articles/PMC3679945/